HSPC117 deficiency in cloned embryos causes placental abnormality and fetal death

Somatic cell nuclear transfer (SCNT) has been successfully used in many species to produce live cloned offspring, albeit with low efficiency. The low frequency of successful development has usually been ascribed to incomplete or inappropriate reprogramming of the transferred nuclear genome. Elucidating the genetic differences between normal fertilized and cloned embryos is key to understand the low efficiency of SCNT. Here, we show that expression of HSPC117, which encodes a hypothetical protein of unknown function, was absent or very low in cloned mouse blastocysts. To investigate the role of HSPC117 in embryo development, we knocked-down this gene in normal fertilized embryos using RNA interference. We assessed the post-implantation survival of HSPC117 knock-down embryos at 3 stages: E9 (prior to placenta formation); E12 (after the placenta was fully functional) and E19 (post-natal). Our results show that, although siRNA-treated in vivo fertilized/produced (IVP) embryos could develop to the blastocyst stage and implanted without any difference from control embryos, the knock-down embryos showed substantial fetal death, accompanied by placental blood clotting, at E12. Furthermore, comparison of HSPC117 expression in placentas of nuclear transfer (NT), intracytoplasmic sperm injection (ICSI) and IVP embryos confirmed that HSPC117 deficiency correlates well with failures in embryo development: all NT embryos with a fetus, as well as IVP and ICSI embryos, had normal placental HSPC117 expression while those NT embryos showing reduced or no expression of HSPC117 failed to form a fetus. In conclusion, we show that HSPC117 is an important gene for post-implantation development of embryos, and that HSPC117 deficiency leads to fetal abnormalities after implantation, especially following placental formation. We suggest that defects in HSPC117 expression may be an important contributing factor to loss of cloned NT embryos in vivo. (C) 2010 Elsevier Inc. All rights reserved.

Localization and distribution of tissue type and urokinase type plasminogen activators and their inhibitors type 1 and 2 in human and rhesus monkey fetal membranes

Fetal membranes consist of 10 distinct layers including components of amnion, chorion and decidua, the latter being of maternal origin. They form mechanically integrated sheets capable of retaining amniotic fluid and play an essential role in protecting fetal growth and development in the pregnant uterus. The extracellular matrix, substrate for plasminogen activators (PAs), is an important supportive framework of the fetal membranes. :Fetal membranes from women with preterm premature rupture of membranes may differ in their protease activity compared with normal membranes. To identify the presence of PAs and their inhibitors (PAI) and their possible role in the process of fetal membrane rupture, this study in investigated the distribution and localization of both protein and mRNA for tissue (t) and urokinase (u) PA and their inhibitors type 1 (PAI-1) and type 2 (PAI-2) in amniochorion of human and rhesus monkey using conventional and. confocal immunofluorescence microscopy. In situ hybridization analysis showed that the distribution and localization of mRNAs for tPA, uPA, PAI-I and PAI-2 were similar in the fetal membranes of human and rhesus monkey; no obvious species difference was observed. Evidence of tPA mRNA was detected in amniotic epithelium, trophoblast cells and nearly all cells of the decidual layer. Strong expression of uPA mRNA was noted in the decidual cells which increased in intensity as the abscission point was approached. Weak staining in chorion laeve trophoblast was also detected. In situ hybridization experiments showed PAI-1 mRNA to be concentrated mainly in the decidual cells, some of which were interposed into the maternal-facing edge of the chorion laeve. Maximal labelling of the decidua occurred towards the zone of abscission. Weak expression of PAI-1 mRNA nas also noted in some cells of the chorion laeve. The distribution of PAI-2 mRNA in amniochorion was also concentrated in the cells of the decidual layer, maximum expression of the mRNA was in the level of abscission. No detectable amount of mRNAs for tPA, uPA, PAI-1 and PAI-2 was found in the fibroblast, reticular and spongy layers. Distribution of the proteins of tPA, uPA and PAI-1 in the fetal membranes of these two species was consistent with the distribution of their mRNA. Anti-PAI-2 immunofluorescence was found to be strongly concentrated in the amniotic epithelium, but PAI-2 mRNA was negative in this layer, suggesting that the epithelium-associated PAI-2 is not of epithelial origin. These findings suggest that a local fibrinolysis in fetal membranes generated by precisely balanced expression of PAs and their inhibitors via paracrine or autocrine mechanisms may play an essential role in fetal membrane development, maturation and in membrane rupture. Following an analysis of the distribution and synthesis of activators and inhibitors it was found that they may play a role in abscission during the third stage of labour. (C) 1998 W. B. Saunders Company Ltd.

Alternative splicing of the FMR1 gene in human tissues of fetal and adult

To study the time- and tissue-specificity of alternative splicing of the FMR1 gene, we analyzed the alternative splicing pattern of the FMR1 gene in human tissues from adult and fetus using RT-PCR coupled with capillary electrophoresis. Seven alternative splicing variants of FMR1 were found in adult liver and lung. The major three alternative splicing variants of the FMR1 gene in all analyzed fetal tissues were same, though the number of minor isoforms and the relative abundance of major isoforms were different. The major difference of the alternative splicing pat tem between adult and fetus was in exon 12 and 17. The results suggest that the alternative splicing pattern of the FMR1 gene is non-tissue-specific in the same developmental stage and a developmental switch may be present.

Serum-free culture of rhesus monkey embryonic stem cells

Previous studies have shown that the maintenance and proliferation of undifferentiated rhesus monkey embryonic stem (rES) cells requires medium supplemented with fetal bovine serum (FBS). Due to the uncharacterized composition and variation in serum nature, the present study aimed to replace the serum-containing medium with a serum-free medium in the rES cell culture. The results showed that after the initial 48-h culture in the routinely used serum-containing medium, rES cells can grow and proliferate for a prolonged period in the serum-free medium composed of DMEM supplemented with a cocktail of BSA, IGF-1, TGF-alpha, bFGF, aFGF, estradiol, and progesterone. rES cells cultured in the serum-free medium maintained high level of alkaline phosphatase activity and OCT4 level. There was no indication of differentiation as judged by the marker gene expression of all three embryonic germ layers and trophoblast. In addition, serum-free culture would not affect the passage capacity and differentiation potential of rES cells. This work will facilitate the future study of induced differentiation of rES cells and other applications.

胎牛血清促进色素上皮衍生因子在皮肤角质形成细胞和成纤维细胞中的表达

下载PDF阅读器目的:探讨表皮角质形成细胞和真皮成纤维细胞中色素上皮衍生因子(PEDF) 表达的影响因素.方法:体外培养角质形成细胞和成纤维细胞,并以10%FBS(胎牛血清)刺激,通过免疫荧光和Western blot检测PEDF的表达.结果:PEDF大多位于细胞浆中,但在细胞核中也有少量表达.细胞浆中PEDF并非均质型分布,而是呈细颗粒状集聚.10%FBS促进角质形成细胞和成纤维细胞中PEDF的集聚和表达,而且这一分布形式不受组胺和佛波肉豆蔻醋酸(PMA) 的影响.结论:10% FBS促进角质形成细胞和成纤维细胞中PEDF的集聚和表达.

人胚胎神经干细胞的分离培养及鉴定研究

目的:研究人胚胎神经干细胞的培养条件及体外分化情况。方法:从12周龄人胚胎脑皮质分离细胞,采用无血清培养技术,协同应用碱性成纤维细胞生长因子(bFGF)、表皮生长因子(EGF)和重组人白血病抑制因子(rhLIF)进行培养;5-溴脱氧尿嘧啶核苷(BrdU)标记检测细胞的增殖能力,间接免疫荧光化学法检测细胞的分化情况。结果:培养得到的大量半悬浮生长的神经干细胞球能够传代培养;BrdU标记阳性,可诱导分化为神经元、星形胶质细胞和少突胶质细胞。结论:人胚胎脑皮质分离细胞培养得到的细胞群具有神经干细胞的基本特征,可进一步用于基础及临床研究。

HORMONAL-REGULATION OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR AND PLASMINOGEN-ACTIVATOR INHIBITOR TYPE-1 IN CULTURED MONKEY SERTOLI CELLS

Sertoli cells play a central role in the control and maintenance of spermatogenesis. Isolated Sertoli cells of mouse and rat testes have been shown to secrete plasminogen activator (PA) and a plasminogen activator inhibitor type-1 (PAI-1) in culture. In this study, we have investigated the hormonal regulation of PA and PAI-1 activities in cultured monkey Sertoli cells. Sertoli cells (5x10(5) cells/well) isolated from infant rhesus monkey testes were preincubated at 35 degrees C for 16 h in 24-well plates precoated with poly(D-lysine) (5 mu g/cm(2)) in 0.5 mi McCoy's 5a medium containing 5% of fetal calf serum and further incubated for 48 h in 0.5 mi serum-free medium with or without various hormones or other compounds, PA as well as PAI-1 activities in the conditioned media were assayed by fibrin overlay and reverse fibrin autography techniques respectively. The Sertoli cells in vitro secreted only tissue-type PA (tPA), no detectable amount of urokinase-type PA (uPA) could be observed, Monkey Sertoli cells were also capable of secreting PAI-1, Immunocytochemical studies indicated that both tPA and PAI-1 positive staining localized in the Sertoli cells, spermatids and residual bodies of the seminiferous epithelium; Northern blot analysis further confirmed the presence of both tPA and PAI-1 mRNA in monkey Sertoli cells. Addition of follicle-stimulating hormone (FSH) or cyclic adenosine monophosphate (cAMP) derivatives or cAMP-generating agents and gonadotrophin-releasing hormone (GnRH) agonist or phorbol ester (PMA) to the cell culture significantly increased tPA activity. PAI-1 activity in the culture was also enhanced by these reagents except 8-bromo-dibutyryl-cAMP, forskolin and 3-isobutyl-1-methylxanthin (MIX) which greatly stimulated tPA activity, whereas decreased PAI-1 activity, implying that neutralization of PAI-1 activity by tile high level of tPA in the conditioned media may occur. These data suggest that increased intracellular signals which activate protein kinase A (PKA), or protein kinase C (PKC) can modulate Sertoli cell tPA and PAI-1 activities, The concomitant induction of PA and PAI-1 by the same reagents in the Sertoli cells may reflect a finely tuned regulatory mechanism in which PAI-1 could limit the excession of the proteolysis.

Establishment, characterization, and virus susceptibility of a new marine cell line from red spotted grouper (Epinephelus akaara)

A marine fish cell line from the snout of red spotted grouper Epinephelus akaara, a protogynous hermaphrodite, was established, characterized, and subcultured with more than 60 passages. The grouper snout cell line (GSC) cells multiplied well in Dulbecco's modified Eagle's medium (DMEM) medium supplemented with 10% fetal bovine serum. The optimal growth temperature was 25 degrees C, and morphologically the cells were fibroblastic. Chromosome analysis revealed that the GSC cell line has a normal diploid karyotype with 2n = 8st + 40t. A virus titration study indicated that the cells were susceptible to turbot Scophthalmus Maximus rhabdovirus (SMRV) (10(8.5) TCID50 ml(-1)), while the viral titer of frog Rana grylio virus 9807 (RGV(9807)) reached 10(3.5) TCID50 ml-1. The infection was confirmed by cytopathic effect (CPE), immunofluorescence, and electron microscopy experiments, which detected the viral particles in the cytoplasm of virus-infected cells, respectively. Further, significant fluorescent signals were observed when the GSC cells were transfected with pEGFP vector DNA, indicating their potential utility for transgenic and genetic manipulation studies.

Total and subcellular distribution of trace elements in the liver of a mother-fetus pair of Dall's porpoises (Phocoenoides dalli)

Total and subcellular hepatic Zn, Cu, Se, Mn, V, Hg, Cd, and Ag were determined in a mother-fetus pair of Dall's porpoises (Phocoenoides dalli). Except for higher fetal Cu concentration, all maternal elements were higher. Elements existed mostly in the cytosol of both animals except in the case of maternal Ag in the microsome and fetal Cu and Ag in the nuclei and mitochondria. In the maternal cytosol, Zn, Mn, Hg, and Ag were present in the high-molecular-weight substances (HMW); Se and V were present in the low-molecular-weight substances (LMW); Cu and Cd were mostly sequestered by metallothionein (MT). In the fetal cytosol, Zn, Se, Mn, Hg, Cd, and Ag were present in the HMW and V in the LMW, while Cu and Ag were mostly associated with MT. MT isoforms were characterized using the HPLC/ICP-MS. Two and four obvious peaks appeared in the maternal and fetal MT fractions, respectively. The highest elemental ion intensities were at a retention time of 7.8 min for the mother, and for the fetus the peak elemental ion intensities occurred at a retention time of 4.3 min, suggesting that different MT isoforms may be involved in elemental accumulation in maternal and fetal hepatocytosols. (C) 2003 Elsevier Ltd. All rights reserved.

Factors affecting the efficiency of somatic cell nuclear transplantation in the fish embryo

Procedures to improve somatic cell nuclear transplantation in fish were evaluated. We reported effects of nonirradiated recipient eggs, inactivated recipient eggs, different combinations between recipient eggs and donor cells, duration of serum starvation, generation number, and passage number of donor cells on developmental rates of nuclear transplant (NT) embryos. Exposure to 25,000 R of gamma-rays inactivated recipient eggs. Single nucleus of cultured, synchronized somatic cell from gynogenetic bighead carp (Aristichthys nobilis) was transplanted into nonirradiated or genetically inactivated unfertilized egg of gibel carp (Carassius auratus gibelio). There was no significant difference in developmental rate between nonirradiated and inactivated recipient eggs (27.27% vs. 25.71%, respectively). Chromosome count showed that 70.59% of NT embryos contained 48 chromosomes. It showed that most NT embryos came from donor nuclei of bighead carp, which was supported by microsatellite analysis of NT embryos. But 23.53% of NT embryos contained more than 48 chromosomes. It was presumed that those superfluous chromosomes came from nonirradiated recipient eggs. Besides, 5.88% of NT embryos were chimeras. Eggs of blunt-snout bream (Megalobrama amblycephala) and gibel carp were better recipient eggs than those of loach (Misgurnus anguillicaudatus) (25% and 18.03% vs. 8.43%). Among different duration of serum starvation, developmental rate of NT embryos from somatic nuclei of three-day serum starvation was the highest, reaching 25.71% compared to 14.14% (control), 20% (five-day), and 21.95% (seven-day). Cultured donor cells of less passage facilitated reprogramming of NT embryos than those of more passage. Recloning might improve the developmental rate of NT embryos from the differentiated donor nuclei. Developmental rate of fourth generation was the highest (54.83%) and the lowest for first generation (14.14%) compared to second generation (38.96%) and third generation (53.01%). (C) 2002 Wiley-Liss, Inc.

Studies on polysaccharides from three edible species of Nostoc (Cyanobacteria) with different colony morphologies: Comparison of monosaccharide compositions and viscosities of polysaccharides from field colonies and suspension cultures

Hot water-soluble polysaccharides woe extracted from field colonies and suspension cultures of Nostoc commune Vaucher, Nostoc flagelliforme Berkeley et Curtis, and Nostoc sphaeroides Kutzing. Excreted extracellular polymeric substances (EPS) were isolated from the media in which the suspension cultures were grown. The main monosaccharides of the field colony polysaccharides from the three species were glucose, xylose, and galactose, with an approximate ratio of 2:1:1. Mannose was also present, but the levels varied among the species, and arabinose appeared only in N. flagelliforme. The compositions of the cellular polysaccharides and EPS from suspension cultures were more complicated than those of the field samples and varied among the different species. The polysaccharides from the cultures of N. flagelliforme had a relatively simple composition consisting of mannose, galactose, glucose, and glucuronic acid, but no xylose, as was found in the field colony polysaccharides. The polysaccharides from cultures of N. sphaeroides contained glucose (the major component), rhamnose, fucose, xylose, mannose, and galactose. These same sugars were present in the polysaccharides from cultures of N. commune, with xylose as the major component. Combined nitrogen in the media had no qualitative influence on the compositions of the cellular polysaccharides but affected those of the EPS of N. commune and N. flagelliforme. The EPS of N. sphaeroides had a very low fetal carbohydrate content and thus was not considered to be polysaccharide in nature. The field colony polysaccharides could be separated by anion exchange chromatography into neutral and acidic fractions having similar sugar compositions. Preliminary linkage analysis showed that 1) xylose, glucose, and galactose were 1-->4 linked, 2) mannose, galactose, and xylose occurred as terminal residues, and 3) branch points occurred in glucose as 1-->3,4 and 1-->3,6 linkages and in xylose as a 1-->3,4 linkage. The polymer preparations from field colonies had higher kinematic viscosities than those from corresponding suspension cultures. The high viscosities of the polymers suggested that they might DE suitable for industrial uses.

Insulin-like growth factor-binding protein-3 plays an important role in regulating pharyngeal skeleton and inner ear formation and differentiation

Insulin-like growth factor-binding protein (IGFBP)-3 is the major insulin-like growth factor (IGF) carrier protein in the bloodstream. IGFBP-3 prolongs the half-life of circulating IGFs and prevents their potential hypo-glycemic effect. IGFBP-3 is also expressed in many peripheral tissues in fetal and adult stages. In vitro, IGFBP-3 can inhibit or potentiate IGF actions and even possesses IGF-independent activities, suggesting that local IGFBP-3 may also have paracrine/autocrine function(s). The in vivo function of IGFBP-3, however, is unclear. In this study, we elucidate the developmental role of IGFBP-3 using the zebrafish model. IGFBP-3 mRNA expression is first detected in the migrating cranial neural crest cells and subsequently in pharyngeal arches in zebrafish embryos. IGFBP-3 mRNA is also persistently expressed in the developing inner ears. To determine the role of IGFBP-3 in these tissues, we ablated the IGFBP-3 gene product using morpholino-modified antisense oligonucleotides (MOs). The IGFBP-3 knocked down embryos had delayed pharyngeal skeleton morphogenesis and greatly reduced pharyngeal cartilage differentiation. Knockdown of IGFBP-3 also significantly decreased inner ear size and disrupted hair cell differentiation and semicircular canal formation. Furthermore, reintroduction of a MO-resistant form of IGFBP-3 "rescued" the MO-induced defects. These findings suggest that IGFBP-3 plays an important role in regulating pharyngeal cartilage and inner car development and growth in zebrafish.